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Gap junctions are small pore-forming protein connections between neighboring cells that mediate the flow of ions and small molecules (≤1 200 Da). As such, they provide a degree of direct cytoplasmic continuity between cells that contributes, along with paracrine pathways, to the intercellular flow of information. These channels are often thought to be long-lived, nonspecific conduits allowing the cell-to-cell spread of ionic currents and small signaling molecules such as cAMP, cGMP, etc. However, in recent years, new evidence has dramatically improved our appreciation of their diversity, regulation, and contribution to normal tissue function and, importantly, has advanced our understanding of the pathogenesis of various diseases. For example, in oncology, the growth and spread of malignant cells in a variety of cancers appears to be dependent on establishing gap junction connections between normal and malignant cells.1,2 In cardiovascular medicine, gap junction alterations have been implicated in the genesis of cardiac arrhythmias,3 remodeling of the arterial wall in hypertension,4–6 and perhaps atherosclerosis.7–9 Indeed, the list goes on, including pathologies in the ophthalmic, neurologic, gastrointestinal, and endocrine systems. Considering their involvement in tissue physiology and pathophysiology, it is essential that we move forward in unraveling the regulatory mechanisms underlying gap junction protein expression and function. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Cowan and his colleagues10 in Toronto and Boston describe an elegant series of experiments demonstrating that the principle gap junction protein in vascular smooth muscle cells, Cx43, is transcriptionally upregulated by hypoxia and stretch, which surprisingly appears to be mediated by the formation of reactive oxygen species (ROS).

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A single gap junction protein consists of two hemi-channels (connexons) on closely opposing cell membranes that dock together to form a single pore-containing conduit enabling permeation by ions, cAMP, cGMP, etc, which affect voltage, pH, and signaling. Each connexon consists of 6 transmembrane monomers termed connexins (Cx), and only a single type of protein is required, and sufficient to form a single gap junction. However, homo-oligomeric as well as hetero-oligomeric connexons are constructed from single connexin monomers, and the cellular complement of connexins can be highly specific, with many cells expressing a restricted repertoire of connexins. Humans have at least 20 different connexins whereas rodents have at least 14 connexins,11 representing a combination of specific gene products and their splice variants. They differ mainly in the C-terminal domain, and all are highly conserved members of a multigene family. The formation of gap junctions is initiated in the ER membrane where the connexins are first synthesized. On transit through the Golgi, they are assembled into mature hexameric connexons which are shuttled to the plasma membrane by vesicular transport, typical of other plasma membrane proteins (Figure). Oddly, their insertion into the plasma membrane appears to be random, ie, not targeted to gap junction domains.12 Once in the membrane, connexons diffuse laterally and cluster into the margins of existing gap junction plaques in the junctional membrane and dock with connexons in neighboring cell membranes to form the final gap junction. Interestingly, recent evidence suggests that connexon hemi-channels, perhaps before the clustering and docking with their counterparts on adjacent cells, can actually open and participate in a variety of cellular processes including the extracellular release of ATP and NAD+ which might contribute paracrine signaling.13

Schematic diagram representing the synthesis and assembly of gap junctions. Initially, local stimuli, in this case hypoxia- or stretch-induced increase in ROS,10 activate transcription of connexin Cx43. The connexins are synthesized in the endoplasmic reticulum (step 1) followed by their oligomerization to connexons (hemi-channels) which occurs between the cis to trans-Golgi network (step 2). Connexons are then transported to the nonjunctional plasma membrane by vesicles, which travel along microtubules and fuse into the membrane bilayer (step 3) where they may be able to open and conduct movement of small signaling molecules. Once in the membrane they diffuse laterally to join the gap junction plaque in the junctional membrane and dock with other connexons on the opposing membranes of neighboring cells. Modified from Ebihara13 with permission.

The roles played by connexins in cell function has recently been addressed in studies directed toward defining diversity versus abundance of specific connexins. For example, Cx43 is expressed in heart and testis as well as numerous other tissues. Not surprisingly, the Cx43 knockout (Cx43K/O), which is devoid of Cx43 protein, is lethal in mice due to severe cardiac malformations.14 However, the replacement of Cx43 with Cx40 using a Cx43 K/O, Cx40 knock-in strategy (ie, “Cx43KI40”) rescues the myocardium and results in viable mice with normal cardiac structure and function.15 However, these mice have hypotrophic testis that produce no sperm. Moreover, many of the Cx43KI40 mice died in the first few weeks of life, and the surviving mice were small and failed to grow normally. These studies suggest clear differences in the functional properties between gap junctions formed from Cx43 and Cx40 connexins; in the heart Cx40 could functionally replace Cx43, but not in the testis or various other tissues. Thus, the concept that gap junctions are functionally interchangeable is not necessarily true. Indeed, the proper compliment of connexin isoforms in any given cell is necessary for normal intercellular communication, and thus, cell and tissue function. Similar conclusions have been drawn from studies in the lens of the eye using this K/O-K/I strategy.16,17

One of the more interesting findings is the observation from experiments in cardiac myocytes that indicate that the half-life of Cx43 in the membrane is on the order of only 90 minutes.18 Moreover, radioactive decay of immunoprecipitated [35S]methionine-labeled Cx43 connexins reveal a monoexponential decay. Thus, not only is Cx43 half-life very short, but there also appears to be no significant pool of longer lived Cx43 connexins, consistent with the idea that the proteins forming cardiac gap junctions are completely turned over several times every day.19 This raises the interesting point that intercellular gap junction networks may be dynamically controlled by short-term, local, regulatory factors. This concept is supported by the recent work of Cowan and colleagues,20 who addressed the regulation of gap junctional proteins in vascular smooth muscle cells (SMCs) and endothelial cells (ECs). For example, they have previously shown that Cx43 gene expression in SMCs is sensitive to mechanical loads, demonstrating that a 20% stretch placed on SMCs in culture transcriptionally increased c-fos mRNA within 30 minutes, Cx43 mRNA (3-fold) within 2 hours and Cx43 protein (7-fold) within 4 to 6 hours. They also demonstrated that the application of shear to ECs maintained in static culture increased Cx43 mRNA levels 4-fold within 1 hour. Notably, Cx37 and Cx40 are the dominant connexins expressed in ECs whereas Cx43 is either not normally expressed or, if so, only at very low levels. This finding supports previous studies showing Cx43 expression in cultured ECs under conditions of disturbed flow,21 and in vivo where Cx43 expression is localized to sites of disturbed flow in rat aorta.22

In this issue, Cowan coworkers20 convincingly demonstrate that hypoxic stimulation (pO2≈15 mm Hg) of rat aortic SMCs reversibly upregulates Cx43 protein within 24 hours. In addition, after 24 hours of hypoxia, Cx43 mRNA levels are elevated, and within 4 hours of reoxygenation begin falling, returning to control levels by 24 hours. That this increase in Cx43 message and protein reflects functional gap junction formation is cleverly demonstrated using fluorescent recovery after photobleaching (FRAP), which monitors real-time diffusion of the photosensitive dye calcein (MW 662) between cells under hypoxic conditions. Like the mRNA and protein data, the FRAP images also show recovery toward control levels with reoxygenation. Culminating this interesting report is their observation that hypoxia, as well as mechanical stretch (20%), stimulates the formation of ROS which are inhibitable by either ebselen or rotenone, both of which also block the upregulation of Cx43 protein, consistent with their suggestion that the hypoxia-induced increase in Cx43 and gap junction formation is mediated by ROS. Because rotenone is a mitochondrial complex 1 inhibitor, they speculate that mitochondrial ROS generation contributes to the regulation of gap junction formation in these cells. While these findings are interesting in their own right, they may have far reaching significance. It has long been appreciated that elevated transmural pressure or hypoxia strongly induce remodeling in various vascular beds. The molecular events involved in the regulation and propagation of the remodelling processes in systemic and pulmonary hypertension are poorly understood, and this report opens new and novel ideas addressing potential sites where interventional therapeutic strategies might focus.

Acknowledgments

The author is supported, in part, by National Institutes of Health grants R01-HL-66273 and R01-HD-40284.